Methods for conjugation of oligosaccharides or polysaccharides to protein carriers through oxime linkages via 3-deoxy-d-manno-octulsonic acid

ABSTRACT

Methods for preparing an oligosaccharide-protein carrier immunogenic conjugate or a polysaccharide-protein carrier immunogenic conjugate. The methods include obtaining an oligosaccharide or polysaccharide having a KDO moiety located at the terminal reducing end of the oligosaccharide or polysaccharide that includes a carbonyl functional group; and reacting the carbonyl functional group of the KDO moiety with an aminooxylated protein carrier molecule resulting in a conjugate that includes a covalent oxime bond between the oligosaccharide and the protein carrier or the polysaccharide and the protein carrier.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/832,448, filed Jul. 21, 2006, which is incorporated herein byreference in its entirety.

FIELD

Disclosed herein are conjugates and methods for making conjugates fromoligosaccharide or polysaccharide antigens.

BACKGROUND

There are numerous human and animal diseases or infections that can becaused by Gram-negative bacteria such as, for example, Bordetella spp.and Haemophilus ducreyi.

Vaccination has proven effective for preventing infection of humans andanimals by Bordetella spp. Killed whole cell and subunit vaccines havebeen used to immunize parenterally to protect humans against pertussiscaused by Bordetella pertussis, a highly contagious, severe respiratoryinfection especially of young children. B. parapertussis causes a milderand less frequent form of the disease, but its incidence and importanceis garnering increasing attention. No vaccine is known to prevent it.Vaccination against B. pertussis does not protect against B.parapertussis. Parapertussis infection followed by pertussis in the sameindividuals has been described in literature. B. pertussis is confinedto human, while B. parapertussis is confined to human and sheep. B.bronchiseptica causes respiratory infections in a variety of hosts:kennel cough in dogs, atrophic rhinitis in piglets, bronchopneumonia inrabbits and guinea pigs. Rarely, it infects humans, but young children,animal handlers and increasingly immuno-compromised individuals aresusceptible. Unlike most bacterial respiratory pathogens, B.bronchiseptica efficiently colonizes the ciliated epithelium of therespiratory tract of the host and may establish chronic infections. Acellular veterinary vaccine is available but it is of limited efficacy.

It has been shown that protection to the infections caused bygram-negative bacteria can be conferred by serum anti-lipopolysaccharide(LPS) IgG. Cohen et al., “Double-blind vaccine-controlled randomisedefficacy trial of an investigational Shigella sonnei conjugate vaccinein young adults,” Lancet 349(9046):155-159, 1997. The LPSes of all threebordetellae share several similar features, though none of them isidentical in structure. B. pertussis produce rough-type LPS comprising aLipid A domain and branched dodecasaccharide chain, carrying unusualsugars and free amino and carboxylic groups. On the basis of SDS-PAGEmigration, it is divided into Band B—Lipid A and a branchednanosaccharide, that if further substituted by a trisaccharide unit istermed Band A. Almost identical core structure was reported for B.bronchiseptica LPS. On the contrary, B. parapertussis core region has asimplified heptasaccharide structure; it does not contain Band Atrisaccharide and Band B lacks one heptose and one N-acetylgalactosaminesubstituents. Only B. bronchiseptica and B. parapertussis synthesizeO-specific polysaccharides (O-SP) and initially it was reported thatthey carry identical structure of linear polymers of 1,4-linked2,3-diacetamido-2,3-dideoxy-α-galactouronic acid (Di Fabio J L et al.,FEMS Microbiol. Lett 97:275-282, 1992). However later, serologicaldifferences between B. bronchiseptica strains were observed and ascribedto the structural variations of the non-reducing end-groups of LPSO-chains (Vinogradov E. et al., Eur. J. Biochem. 276:7230-7236, 2000).As it was reported for Vibrio cholerae O1 serotype Ogawa and Inaba, thenon-reducing end-groups play a significant role as major epitopes inserological reactions (Wang J., J. Biol. Chem. 273:2777-2783, 1998).Similar observation was made in case of Salmonella O40 and O43serotypes.

Chancroid is a sexually transmitted genital ulcer disease (GUD) causedby the bacterium Haemophilus ducreyi. Chancroid presents withcharacteristic and persistent genital ulcers on the external genitals,associated with regional lymphadenopathy in 50% of cases. The disease iscommon in many developing countries, and is considered a significantrisk factor together with other genital GUD, e.g. herpes simplex virus 2(HSV-2) for heterosexual HIV transmission in geographic areas where bothdiseases are prominent.

A number of putative virulence factors of H. ducreyi have been describedwhich may play a role in pathogenicity of this organism. Two of thesefactors are toxins: a hemolytic toxin and cytolethal distending toxin.The outer membrane proteins, DsrA and DltA, have been shown to promoteresistance to killing by normal human serum. The hemoglobin receptorHgbA and the Cu, Zn-superoxide dismutase both seem to play a role iniron acquisition for H. ducreyi. Filamentous hemagglutinin like proteinis involved in inhibition of phagocytosis. Heat-shock proteins (HSP) ofH. ducreyi situated on the surface of the bacteria are responsible forprotection of these bacteria against changes in the environment andenhance H. ducreyi adhesion to mammalian cells. Additionally, a numberof proteins have been shown to play a role in adherence.

The lipooligosaccharide (LOS) produced by H. ducreyi is a putativevirulence facto, as well. Previous studies have shown that LOS plays arole in adherence of bacteria to keratinocytes and human foreskinfibroblasts and also contribute to the development of lesions in animalmodels. Structural studies have been performed on the LOS from a numberof H. ducreyi strains, e.g. 35000, ITMA 2665, 3147, 5535, CCUG 7470,4438 and others. These studies have shown that the predominant form ofthe core oligosaccharide of the LOS is composed of 10 saccharides with alactosamine or sialyllactosamine at the non-reducing end and isexpressed by majority of strains.

H. ducreyi enters the skin or mucosa through wounds and attaches toextracellular matrix and to cells. This stimulates an inflammatoryresponse with the development of pro-inflammatory cytokines and assemblyof phagocytic cells; granulocytes and macrophages, at the infectionsite. H. ducreyi may be found both intra and extracellularly. Theinflammatory process may clear the organism partially but may also causetissue destruction and chronic infection with granuloma formation asobserved in rabbit model of infection.

The mediators of immunity to chancroid are not known. Data from patientsand infected volunteers indicate that this local infection does notconfer immunity against subsequent re-infection and do not induce anantibody response. The results from these experiments indicate that thecytokine pattern and the type of cells involved in the early immuneresponse to H. ducreyi, may have features of a Th1 response, including apoor or no antibody response. The in vitro studies of interactions of H.ducreyi with human monocyte-derived-dendritic cells and with macrophagesconfirmed an initial Th1 response. Studies in a rabbit model showed thatboth antibodies and cellular immunity contributed to reducing the numberof bacteria in the lesions, thus contributing to protection. Data from aswine model indicated that antibodies alone, at levels achieved onlyafter more than 3 injections of live bacteria, are sufficient forprotection. Antibodies to different bacterial cell components weredetected in the late stage of disease in sera from patients withchancroid, but antibodies neutralizing CDT have been detected only inabout 28% of chancroid cases. It has also been noted that antibodiesspecific to the LOS of this organism enhance opsonophagocytic killing ofH. ducreyi in vitro, but such antibodies are not elicited in sufficientamounts after repeated dermal injections of bacteria to animals. Lowlevel of induced LOS antibodies may be due to the fact that the LOSstructure resembles terminal saccharides of paraglobise, a major antigenon human erythrocytes and muscles. Since re-infection with H. ducreyican occur, the immunity, including the amount and specificity ofantibodies elicited by this local infection, is likely not sufficientfor protection.

The covalent binding of oligosaccharide to carrier proteins by randomactivation of the saccharide using CDAP and ADH as the linker, resultedin conjugates that induced higher levels of IgG anti LOS than repeatedinjections of the whole cell (Lundquist A, Ahlman k T. Lagergärd,“Preparation and immunological properties of Haemophilus ducreyilipooligosaccharide-protein conjugates,” ASM Meeting, abstract e-044,New Orleans, 2004). A vaccine to prevent chancroid would reduce/preventthe disease burden and have the added benefit of reducing HIV incidence.

Shigellae are Gram-negative bacteria, pathogens to humans only, that cancause endemic and epidemic dysentery worldwide, especially in thedeveloping countries. The symptoms usually start with watery diarrheathat later develops into dysentery, characterized by high fever, bloodand mucus in the stool, and cramps. Shigella flexneri causes dysenterymostly in developing countries with more fatalities then any otherShigella species. The disease can be prevented by vaccination using thepolysaccharide part of the LPS as an immunogen.

SUMMARY

Disclosed herein are methods for preparing an oligosaccharide-proteincarrier immunogenic conjugate or a polysaccharide-protein carrierimmunogenic conjugate. The methods include:

obtaining an oligosaccharide or polysaccharide having an anhydro3-deoxy-D-manno-octulsonic acid moiety located at the terminal reducingend of the oligosaccharide or polysaccharide that includes a carbonylfunctional group; and

reacting the carbonyl functional group of the anhydro3-deoxy-D-manno-octulsonic acid moiety with an aminooxylated proteincarrier molecule resulting in an oligosaccharide-protein carrierimmunogenic conjugate or polysaccharide-protein carrier immunogenicconjugate that includes a covalent oxime bond between theoligosaccharide and the protein carrier or the polysaccharide and theprotein carrier.

Also described herein are immunogenic conjugates comprising thestructure of:

Pr—Sp—O—N═C(COOH)—anh-KDO—OS

wherein Pr is a carrier protein, Sp is an optional spacer moiety,anh-KDO is an anhydro moiety from 3-deoxy-D-manno-octulsonic acid, andOS is an oligosaccharide or polysaccharide residue from the cleavage ofLipid A from a lipopolysaccharide.

Further disclosed are methods of eliciting an immune response in asubject, comprising administering to the subject the above-describedconjugates, thereby eliciting an immune response in the subject.

Another embodiment for preparing an oligosaccharide-protein carrierimmunogenic conjugate or polysaccharide-protein carrier immunogenicconjugate includes obtaining an oligosaccharide or polysaccharide havingan anhydro 3-deoxy-D-manno-octulsonic acid moiety located at theterminal reducing end of the oligosaccharide or polysaccharide. Theanhydro 3-deoxy-D-manno-octulsonic acid moiety of the oligosaccharide orpolysaccharide is reacted with a heterobifunctional compound thatincludes at least one aminooxy group. Subsequently, the resultingfunctionalized oligosaccharide or polysaccharide is reacted with aprotein carrier to produce an oligosaccharide-protein carrierimmunogenic conjugate or polysaccharide-protein carrier immunogenicconjugate that includes a covalent oxime bond between theoligosaccharide and the protein carrier or the polysaccharide and theprotein carrier.

The foregoing and other features and advantages will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF TILE DRAWINGS

FIG. 1 is a conjugation reaction scheme that depicts (a) the synthesisof aminooxylated protein, (b) the synthesis of an oligosaccharide orpolysaccharide that includes a carbonyl functional group (i.e, ketone),and (c) the conjugation of the aminooxylated protein with thecarbonyl-functional oligosaccharide or polysaccharide. Pr is a carrierprotein, LPS is lipopolysaccharide, LOS is a lipooligosaccharide,O-chain is an O-antigen oligosaccharide or polysaccharide chain, Core isa core oligosaccharide or polysaccharide chain, KDO4P is3-deoxy-D-manno-octulsonic acid moiety phosphorylated at position C4,and anhydro-KDO is described below.

FIG. 2 is a LPS structure of Bordetella parapertussis and Bordetellabronchiseptica. A novel pentasaccharide(-4-β-ManNAc3ANcAN-4-β-GlcNAc3NAcAN-4-α-GalNAc-4-β-ManNAc3NAcA-3-β-FucNAc4NMe-)present between the O-SP and the core was identified. In addition,besides the reported structure the O-SP of B. bronchiseptica and B.parapertussis being a homopolymer of 1,4-linked2,3-diacetamido-2,3-dideoxy-α-galacturonic acid, it was found that bothO-SP contain amidated uronic acids, the number of which varied betweenstrains (Preston et al., J. Biol. Chem., 2006 (in press)). Certainfragments (-6-β-GlcNAc-4-β-ManNAc3NAcA-3-β-FucNAc4NMe-) are not presentor partially present (residues with *) in B. parapertussis. Two types ofO-SP end groups (Vinogradov et al., Eur. J. Biochem. 267:7230-7237,2000) (A) were found in B. bronchiseptica and only one, Ala-type, in B.parapertussis.

FIG. 3 is a MALDI-TOF spectrum of BSA-ONH₂/BbRb50 (conjugate #2 in Table2 below).

FIG. 4 is a MALDI-TOF spectrum of BSA-ONH₂/OS (H. ducreyi) (conjugate #1in Table 4 below).

FIG. 5 is a MALDI-TOF spectrum of TT-ONH₂/OS (H. ducreyi) (conjugate #2in Table 4 below).

FIG. 6 is an ESI-MS spectrum of B. pertussis OS used for conjugation inExample 3.

FIG. 7 is an ESI-MS spectrum for B. bronchiseptica OS-core used forconjugation in Example 3.

FIG. 8 is an SDS-PAGE gel result showing an increase in molecular sizeof BSA-ONH₂ /S. flexnerii 2a O-SP conjugate (line 3) over BSA-ONH₂ (line2). Line 1 is a marker. 10% NUPAGE MES gel was used in this experiment.The highest marker line corresponds to 188 kDa.

DETAILED DESCRIPTION I. Abbreviations

-   -   ADH: adipic acid dihydrazide    -   AT: anthrax toxin    -   ATR: anthrax toxin receptor    -   EDAC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl    -   EF: edema factor    -   GLC-MS: gas-liquid chromatography-mass spectrometry    -   kDa: kilodaltons    -   LC-MS: liquid chromatography-mass spectrometry    -   LeTx: lethal toxin    -   LF: lethal factor    -   LOS: lipooligosaccharide    -   LPS: lipopolysaccharide    -   MALDI-TOF: matrix-assisted laser desorption ionization        time-of-flight    -   OS: oligosaccharide    -   μg: microgram    -   μl: microliter    -   PA: protective antigen    -   PBS: phosphate buffered saline    -   SBAP: succinimidyl 3-(bromoacetamido) propionate    -   SFB: succinimidylformylbenzoate    -   SPDP: N-hydroxysuccinimide ester of 3-(2-pyridyl        dithio)-propionic acid    -   SLV: succinimidyllevulinate    -   TT: tetanus toxoid        The saccharide units disclosed herein are abbreviated as below        following conventional oligosaccharide/polysaccharide        nomenclature:    -   anhKDO: anhydro KDO    -   Fuc: fucose    -   Gal: galactose    -   Glc: glucose,    -   GlcNAc: N-acetylglucosamine    -   GalNAc: N-acetylgalactosamine    -   Hep: glycero-D-manno-heptopyranoside (heptose)    -   Hex: hexose    -   Man: mannose    -   NeuNAc: N-acetylneuramic acid

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 2000 (ISBN 019879276X); Kendrew a al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Publishers, 1994 (ISBN0632021829); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by Wiley, John& Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. Also, as used herein, the term “comprises” means“includes.” Hence “comprising A or B” means including A, B, or A and B.It is further to be understood that all nucleotide sizes or amino acidsizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides or other compounds are approximate, andare provided for description. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present disclosure, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingexplanations of terms, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

In order to facilitate review of the various examples of thisdisclosure, the following explanations of specific terms are provided:

Adjuvant: A substance that non-specifically enhances the immune responseto an antigen. Development of vaccine adjuvants for use in humans isreviewed in Singh et al. (Nat. Biotechnol. 17:1075-1081, 1999), whichdiscloses that, at the time of its publication, aluminum salts, such asaluminum hydroxide (Amphogel, Wyeth Laboratories, Madison, N.J.), andthe MF59 microemulsion are the only vaccine adjuvants approved for humanuse. An aluminum hydrogel (available from Brentg Biosector, Copenhagen,Denmark, is another common adjuvant).

In one embodiment, an adjuvant includes a DNA motif that stimulatesimmune activation, for example the innate immune response or theadaptive immune response by T-cells, B-cells, monocytes, dendriticcells, and natural killer cells. Specific, non-limiting examples of aDNA motif that stimulates immune activation include CpGoligodeoxynucleotides, as described in U.S. Pat. Nos. 6,194,388;6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and6,429,199.

Analog, Derivative or Mimetic: An analog is a molecule that differs inchemical structure from a parent compound, for example a homolog(differing by an increment in the chemical structure, such as adifference in the length of an alkyl chain), a molecular fragment, astructure that differs by one or more functional groups, a change inionization. Structural analogs are often found using quantitativestructure activity relationships (QSAR), with techniques such as thosedisclosed in Remington (The Science and Practice of Pharmacology, 19thEdition (1995), chapter 28). A derivative is a biologically activemolecule derived from the base structure. A mimetic is a molecule thatmimics the activity of another molecule, such as a biologically activemolecule. Biologically active molecules can include chemical structuresthat mimic the biological activities of a compound.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, humans, non-human primates,dogs, cats, horses, and cows.

Antibody: A protein (or protein complex) that includes one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is generally atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” (about 50-70 kDa) chain. The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

Antibodies for use in the methods and devices of this disclosure can bemonoclonal or polyclonal. Merely by way of example, monoclonalantibodies can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein (Nature 256:495-97, 1975) orderivative methods thereof. Detailed procedures for monoclonal antibodyproduction are described in Harlow and Lane, Using Antibodies: ALaboratory Manual, CSHL, New York, 1999.

Antigen: A compound, composition, or substance that may be specificallybound by the products of specific humoral or cellular immunity, such asan antibody molecule or T-cell receptor. Antigens can be any type ofbiologic molecule including, for example, simple intermediarymetabolites, sugars (e.g., oligosaccharides), lipids, and hormones aswell as macromolecules such as complex carbohydrates (e.g.,polysaccharides), phospholipids, nucleic acids and proteins. Commoncategories of antigens include, but are not limited to, viral antigens,bacterial antigens, fungal antigens, protozoa and other parasiticantigens, tumor antigens, antigens involved in autoimmune disease,allergy and graft rejection, toxins, and other miscellaneous antigens.In one example, an antigen is a lipopolysaccharide antigen.

Carrier: An immunogenic molecule to which an antigen such as anoligosaccharide or polysaccharide can be bound. When bound to a carrier,the bound molecule may become more immunogenic. Carriers are chosen toincrease the immunogenicity of the bound molecule and/or to elicitantibodies against the carrier which are diagnostically, analytically,and/or therapeutically beneficial. Covalent linking of a molecule to acarrier confers enhanced immunogenicity and T-cell dependence (Pozsgayet al., PNAS 96:5194-97, 1999; Lee et al., J. Immunol. 116:1711-18,1976; Dintzis et al., PNAS 73:3671-75, 1976). Useful carriers includepolymeric carriers, which can be natural (for example, proteins frombacteria or viruses), semi-synthetic or synthetic materials containingone or more functional groups to which a reactant moiety can beattached.

Examples of bacterial products for use as carriers include bacterialtoxins, such as B. anthracis PA (including fragments that contain atleast one antigenic epitope and analogs or derivatives capable ofeliciting an immune response), LF and LeTx, and other bacterial toxinsand toxoids, such as tetanus toxin/toxoid, diphtheria toxin/toxoid, P.aeruginosa exotoxin/toxoid/, pertussis toxin/toxoid, and C. perfringensexotoxin/toxoid. Viral proteins, such as hepatitis B surface antigen andcore antigen can also be used as carriers.

Covalent Bond: An interatomic bond between two atoms, characterized bythe sharing of one or more pairs of electrons by the atoms. The terms“covalently bound” or “covalently linked” refer to making two separatemolecules into one contiguous molecule. The terms include reference tojoining a hapten or antigen indirectly to a carrier molecule, with anintervening linker molecule.

Epitope: An antigenic determinant. These are particular chemical groupsor contiguous or non-contiguous peptide sequences or saccharide units ona molecule that are antigenic, that is, that elicit a specific immuneresponse. An antibody binds a particular antigenic epitope based on thethree dimensional structure of the antibody and the matching (orcognate) epitope.

Immune Response: A response of a cell of the immune system, such as aB-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus. Animmune response can include any cell of the body involved in a hostdefense response, for example, an epithelial cell that secretesinterferon or a cytokine. An immune response includes, but is notlimited to, an innate immune response or inflammation.

Immunogenic Conjugate or Composition: A term used herein to mean acomposition useful for stimulating or eliciting a specific immuneresponse (or immunogenic response) in a vertebrate. In some embodiments,the immunogenic response is protective or provides protective immunity,in that it enables the vertebrate animal to better resist infection ordisease progression from the organism against which the immunogeniccomposition is directed. One specific example of a type of immunogeniccomposition is a vaccine.

Immunogen: A compound, composition, or substance which is capable, underappropriate conditions, of stimulating the production of antibodies or aT-cell response in an animal, including compositions that are injectedor absorbed into an animal.

Immunologically Effective Dose: An immunologically effective dose of theoligosaccharide-protein or polysaccharide-protein conjugates of thedisclosure is therapeutically effective and will prevent, treat, lessen,or attenuate the severity, extent or duration of a disease or condition,for example, infection by Bordetella parapertussis or Bordetellabronchiseptica.

Inhibiting or Treating a Disease: inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as respiratory tract infections. “Treatment” refers to atherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition after it has begun to develop. As used herein,the term “ameliorating,” with reference to a disease, pathologicalcondition or symptom, refers to any observable beneficial effect of thetreatment. The beneficial effect can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease, a slower progression of the disease, a reduction in the numberof relapses of the disease, an improvement in the overall health orwell-being of the subject, or by other parameters well known in the artthat are specific to the particular disease.

Isolated: An “isolated” biological component (such as alipopolysaccharide) has been substantially separated or purified awayfrom other biological components in the cell of the organism in whichthe component naturally occurs, such as other chromosomal andextra-chromosomal DNA and RNA, proteins, glycolipids and organelles.

Lipopolysaccharide (LPS): LPS is an endotoxin that is a majorsuprastructure of the outer membrane of Gram-negative bacteria whichcontributes greatly to the structural integrity of the bacteria, andprotects them from host immune defenses. LPS typically contains threecomponents: (a) Lipid A (a hydrophobic domain that typically consists ofa glucosamine disaccharide that is substituted with phosphate groups andlong chain fatty acids in ester and amide linkages); (b) a corepolysaccharide or oligosaccharide that can include, for example,heptose, glucose, galactose and N-acetylglucosamine units depending uponthe genera and species of bacteria; and (c) optionally, polysaccharidedistal or side chain(s) (often referred to as the “0 antigen” that caninclude, for example, mannose, galactose, D-glucose,N-acetylgalactosamine, N-acetylglucosamine, L-rhamnose, and adideoxyhexose depending upon the genera and species of bacteria). LipidA and the core polysaccharide or oligosaccharide domains are joinedtogether by one or more units of 3-deoxy-D-manno-octulsonic acid (“KDO”,also known as ketodeoxyoctonate). A lipooligosaccharide (LOS) (alsoknown as a “short chain LPS”) commonly refers to an LPS that containsLipid A plus a core polysaccharide or oligosaccharide (e.g., in H.ducreyi and B. pertussis that does not naturally contain any 0 antigenchains). As used herein, the term LPS can include short chain LPS andLOS.

Oligosaccharide (OS): As used herein, the term “oligosaccharide” is notnecessarily restricted to a molecule having a specific number ofsaccharide units. However, in general, an oligosaccharide is acarbohydrate that contains from about 3 to about 10 simple sugars (e.g.,monosaccharides) linked together. O-specific oligosaccharide (O-SP)refers to an O-specific oligosaccharide chain attached to a coreoligosaccharide or polysaccharide chain. The oligosaccharides orpolysaccharides conjugated to the protein carrier do not include a lipidcomponent.

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic compounds or molecules, such as one or more SARS-CoV nucleicacid molecules, proteins or antibodies that bind these proteins, andadditional pharmaceutical agents. The term “pharmaceutically acceptablecarrier” should be distinguished from “carrier” as described above inconnection with a hapten/carrier conjugate or an antigen/carrierconjugate.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used. The terms “polypeptide” or “protein” as used herein areintended to encompass any amino acid sequence and include modifiedsequences such as glycoproteins. The term “polypeptide” is specificallyintended to cover naturally occurring proteins, as well as those whichare recombinantly or synthetically produced.

The term “residue” or “amino acid residue” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.

Protein: A molecule, particularly a polypeptide, comprised of aminoacids.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purifiedpeptide, protein, conjugate, LPS, or other active compound is one thatis isolated in whole or in part from proteins, lipids or othercontaminants. Generally, substantially purified peptides, proteins,conjugates, LPSs or other active compounds for use within the disclosurecomprise more than 80% of all macromolecular species present in apreparation prior to admixture or formulation of the peptide, protein,conjugate, LPS or other active compound with a pharmaceutical carrier,excipient, buffer, absorption enhancing agent, stabilizer, preservative,adjuvant or other co-ingredient in a complete pharmaceutical formulationfor therapeutic administration. More typically, the peptide, protein,conjugate, LPS or other active compound is purified to represent greaterthan 90%, often greater than 95% of all macromolecular species presentin a purified preparation prior to admixture with other formulationingredients. In other cases, the purified preparation may be essentiallyhomogeneous, wherein other macromolecular species are not detectable byconventional techniques.

Therapeutically Effective Amount: A quantity of a specified agentsufficient to achieve a desired effect in a subject being treated withthat agent. For example, this may be the amount of an OS-protein orpolysaccharide-protein conjugate useful in increasing resistance to,preventing, ameliorating, and/or treating infection and disease causedby Bordetella parapertussis, Bordetella bronchiseptica, Haemophilusducreyi, Bordetella pertussis, Vibrio cholere, or Haemophilus influenzainfection in a subject. Ideally, a therapeutically effective amount ofan agent is an amount sufficient to increase resistance to, prevent,ameliorate, and/or treat infection and disease caused by Bordetellaparapertussis, Bordetella bronchiseptica, Haemophilus ducreyi,Bordetella pertussis, Vibrio cholere, or Haemophilus influenza infectionin a subject without causing a substantial cytotoxic effect in thesubject. The effective amount of an agent useful for increasingresistance to, preventing, ameliorating, and/or treating infection anddisease caused by Bordetella parapertussis, Bordetella bronchiseptica,Haemophilus ducreyi, Bordetella pertussis, Vibrio cholere, orHaemophilus influenza infection in a subject will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition.

Toxoid: A nontoxic derivative of a bacterial exotoxin produced, forexample, by formaldehyde or other chemical treatment. Toxoids are usefulin the formulation of immunogenic compositions because they retain mostof the antigenic properties of the toxins from which they were derived.

Vaccine: A vaccine is a pharmaceutical composition that elicits aprophylactic or therapeutic immune response in a subject. In some cases,the immune response is a protective response. Typically, a vaccineelicits an antigen-specific immune response to an antigen of a pathogen,for example, a bacterial or viral pathogen, or to a cellular constituentcorrelated with a pathological condition. A vaccine may include apolynucleotide, a peptide or polypeptide, a polysaccharide, a virus, abacteria, a cell or one or more cellular constituents. In some cases,the virus, bacteria or cell may be inactivated or attenuated to preventor reduce the likelihood of infection, while maintaining theimmunogenicity of the vaccine constituent.

As described above, disclosed herein are methods for conjugatingoligosaccharides or polysaccharides having a 3-deoxy-D-manno-octulsonicacid moiety located at the terminal reducing end of the oligosaccharidesor polysaccharides. According to the methods, binding the OS by KDO atthe reducing end of the OS means that all of the conserved OS structureremains intact or unmodified (e.g., none of the saccharide residues areoxidized) which provides more potential sites for interaction leading tohigher immunogenicity. The conjugates disclosed herein preserve theexternal non-reducing end of the OS, are recognized by antisera, andinduce immune responses in mice.

The oligosaccharide may be obtained from gram-negative bacteria such asHaemophilus ducreyi, Bordetella bronchiseptica, Bordetellaparapertussis, Bordetella pertussis, Vibrio cholere, or Haemophilusinfluenza or any other Haemophilus spp. These bacteria typically containa lipopolysaccharide (LPS) with a 3-deoxy-D-manno-octulsonic acid moietyphosphorylated at position C4 on the 3-deoxy-D-manno-octulsonic acidmoiety.

Also disclosed herein are novel techniques for binding S. flexnerii O-SPto protein carrier via KDO. This technique can be applied to otherShigellae like S. dysenteriae and S. sonnei, as well as to otherenterobacteriacea and other gram-negative bacteria having KDO moleculebetween Lipid A and oligo/polysaccharide chain of their LPS.

The target oligosaccharides or polysaccharides for conjugation typicallyare those that carry epitopes in their structure. Examples of sucholigosaccharides or polysaccharides are described below in more detailin examples 1 and 2. The oligosaccharides or polysaccharides that areconjugated include a general structure of:

O-chain (if present)-core OS-anhydro-KDO

The anhydro-KDO moiety is the moiety that results after acid hydrolysistreatment of the isolated LOS or LPS as described in more detail belowand it has a structure represented by (anhydro-KDO could also bereferred to as 4, 8(7)-anhydro derivative of KDO):

The oligosaccharide or polysaccharide typically is derived from LPSpresent in the bacteria identified above. The LPS initially is isolatedfrom the other constituents of the bacteria cell structure. IllustrativeLPS-isolation techniques are described, for example, in Westphal et al.,Meth. Carbohydr. Chem. 5:83-89, 1965, which is incorporated herein byreference in its entirety, and typically involve isolation orpurification via a phenol-water extraction. Other LPS-isolationtechniques include enzyme digestion and alcohol precipitation,chromatography by gel filtration and ion-exchange.

The isolated LPS then is subjected to mild acid hydrolysis to cleave theLipid A from the polysaccharide or oligosaccharide domain such that the3-deoxy-D-manno-octulsonic acid remains linked to the polysaccharide oroligosaccharide domain. Such techniques are described, for example, inAuzanneau, J. Chem. Soc. Perkin Trans. 1:509-516, 1991 and Rybka et al.,J. Microblol. Methods 64(2):171-184, 2006, both of which areincorporated herein by reference. Illustrative hydrolysis conditionsinclude treating the LPS with acetic acid for 1-3 hours at about 100°C., or hydrolyzing LPS in a mixture of acetic acid and sodium acetate(e.g., treating 50 mg LPS with a mixture of 73.5 ml of 0.2 M acetic acidand 26.5 ml of 0.2 M sodium acetate for 5 hours at 100° C. in 5 mlvolume). The acid hydrolysis transforms the KDO structure in theisolated LPS to an anhydro-KDO structure.

Conjugation of the oligosaccharide or polysaccharide to the carrierprotein is accomplished via formation of an oxime linkage between acarbonyl functional group present in the KDO moiety and an aminooxyfunctional group present on the carrier protein. The oxime linkagereaction is a chemoselective ligation since it involves the aqueouscovalent coupling of unprotected, highly functionalized biomoleculesthat contain at least a pair of functional groups that react togetherexclusively, within a biological environment. Oxime linkages can beformed in an aqueous reaction environment, and are stable, from pH 5 topH 7. Other advantageous features of forming oxime linkages include arelatively short reaction time, a good yield, and formation at ambienttemperature. These conditions avoid denaturation of the carrier protein.

The reactive carbonyl functional group present in the KDO moiety can bean aldehyde or a ketone remaining after acid hydrolysis cleavage of theLipid A from the LPS. The carrier protein is functionalized with anaminooxy group. The synthetic scheme for forming the oxime linkage isshown below:

Pr—Sp—O—NH₂+HOOC—C(O)—anh-KDO—OS→Pr—Sp—O—N═C(COOH)—anh-KDO—OS

wherein Pr is a carrier protein, Sp is an optional spacer moiety,anh-KDO is anhydro-KDO, and OS is an oligosaccharide or polysaccharideresidue from the cleavage of Lipid A from LPS. Condensation between thecarbonyl and aminooxy groups leads to a stable oxime linkage between theOS and carrier protein. The spacer moiety may have any structure that ispresent in the linker reagents as described below. Alternatively, theHOOC—C(O)—anh-KDO—OS structure could be reacted initially with anaminooxy reagent, and the resulting aminooxy-functionalized reactantcould be reacted with the protein.

The oxime conjugation reaction is performed at pH 5 to about pH 7 atambient temperature conditions in an aqueous environment. The reactiontime typically ranges from about 8 to about 24 hours. However, less than100% conjugation completion can be achieved in less than 8 hours, andthe 8-24 hour reaction time assumes near 100% conjugation completion.

The carrier protein (or anh-KDO—OS) can be functionalized to include atleast one reactive aminooxy moiety by various techniques as described,for example, in Kielb et al., J. Org. Chem. 70:6987-6990, 2005 and U.S.Patent Application Publication No. 2005/0169941, both of which areincorporated herein by reference. Functionalization of the carrierprotein can result in the inclusion of an optional spacer moiety asnoted above. In illustrative examples, a carrier protein (or anh-KDO—OS)may be reacted with a linker reagent to incorporate the spacer moietyand the aminooxy functional moiety. The linker reagent typically is aheterobifunctional compound that includes at least one aminooxy groupand a second functional group that is reactive with the carrier protein.Suitable linker reagents include aminooxy-thiol compounds. Illustrativeaminooxy-thiol linker reagents include aminoooxy-alkyl-thiols such as(thiolalkyl)hydroxylamines (e.g., O-(3-thiolpropyl)hydroxylamine) andaminooxy-aryl-thiols. In the case of aminooxy-thiol linker reagents, thecarrier protein may be treated to introduce thiol-reactive groups. Forexample, the carrier protein may be treated with a treatment agent thatintroduces thiol-reactive haloacetamido or thiol-reactive maleimidomoieties onto the carrier protein. The haloacetamido-containing proteinor maleimido-containing protein is reacted with the aminooxy-thiolreagent to form the aminooxylated carrier protein via the formation ofstable thioether linkages.

The amount of oligosaccharide or polysaccharide reacted with the amountof protein may vary depending upon the specific LPS from which the OS isderived and the carrier protein. However, the respective amounts shouldbe sufficient to introduce about 5-20 chains of OS (PS) onto theprotein. In certain examples, the mol ratio of carbonyl groups on OS(PS) to aminooxy groups on the protein may range from about 0.3:1 toabout 1:3, more particularly 1:1 to about 1:2, and more preferably about1:1. The resulting number of oligosaccharide chains bound to a singleprotein carrier molecule may vary depending upon the specific LPS andthe carrier protein, but in general, about 5 to about 20, morepreferably about 10, OS chains can be bound to each protein carriermolecule. The yield based on the amount of protein ranges from about 70to about 90% in protein derivatization step and about 70 to about 90%after the conjugation with the OS.

Specific, non-limiting examples of water soluble protein carriersinclude, but are not limited to, natural, semi-synthetic or syntheticpolypeptides or proteins from bacteria or viruses. In one embodiment,bacterial products for use as carriers include bacterial wall proteinsand other products (for example, streptococcal or staphylococcal cellwalls), and soluble antigens of bacteria. In another embodiment,bacterial products for use as carriers include bacterial toxins.Bacterial toxins include bacterial products that mediate toxic effects,inflammatory responses, stress, shock, chronic sequelae, or mortality ina susceptible host. Specific, non-limiting examples of bacterial toxinsinclude, but are not limited to: B. anthracis PA (for example, asencoded by bases 143779 to 146073 of GenBank Accession No. NC 007322,herein incorporated by reference), including variants that share atleast 90%, at least 95%, or at least 98% amino acid sequence homology toPA, fragments that contain at least one antigenic epitope, and analogsor derivatives capable of eliciting an immune response; B. anthracis LF(for example, as encoded by the complement of bases 149357 to 151786 ofGenBank Accession No. NC 007322); bacterial toxins and toxoids, such astetanus toxin/toxoid (for example, as described in U.S. Pat. Nos.5,601,826 and 6,696,065); diphtheria toxin/toxoid (for example, asdescribed in U.S. Pat. Nos. 4,709,017 and 6,696,065); P. aeruginosaexotoxin/toxoid/(for example, as described in U.S. Pat. Nos. 4,428,931,4,488,991 and 5,602,095); pertussis toxin/toxoid (for example, asdescribed in U.S. Pat. Nos. 4,997,915, 6,399,076 and 6,696,065); and C.perfringens exotoxin/toxoid (for example, as described in U.S. Pat. Nos.5,817,317 and 6,403,094). Viral proteins, such as hepatitis B surfaceantigen (for example, as described in U.S. Pat. Nos. 5,151,023 and6,013,264) and core antigen (for example, as described in U.S. Pat. Nos.4,547,367 and 4,547,368) can also be used as carriers, as well asproteins from higher organisms such as keyhole limpet hemocyanin,horseshoe crab hemocyanin, edestin, mammalian serum albumins, andmammalian immunoglobulins.

Following conjugation of the oligosaccharide or polysaccharide to thecarrier protein, the conjugate can be purified by a variety oftechniques well known to one of skill in the art. One goal of thepurification step is to remove the unbound oligosaccharide orpolysaccharide from the conjugation reaction product composition. Onemethod for purification, involving ultrafiltration in the presence ofammonium sulfate, is described in U.S. Pat. No. 6,146,902.Alternatively, the conjugates can be purified away from unreactedoligosaccharide/polysaccharide and carrier by any number of standardtechniques including, for example, size exclusion chromatography,density gradient centrifugation, hydrophobic interaction chromatography,or ammonium sulfate fractionation. See, for example, Anderson et al., J.Immunol. 137:1181-1186, 1986 and Jennings & Lugowski, J. Immunol.127:1011-1018, 1981. The compositions and purity of the conjugates canbe determined by GLC-MS and MALDI-TOF spectrometry.

The conjugates disclosed herein may be included in pharmaceuticalcompositions (including therapeutic and prophylactic formulations),typically combined together with one or more pharmaceutically acceptablevehicles and, optionally, other therapeutic ingredients (for example,antibiotics or anti-inflammatories).

Such pharmaceutical compositions can be administered to subjects by avariety of mucosal administration modes, including by oral, rectal,intranasal, intrapulmonary, or transdermal delivery, or by topicaldelivery to other surfaces. Optionally, the conjugate can beadministered by non-mucosal routes, including by intramuscular,subcutaneous, intravenous, intra-atrial, intra-articular,intraperitoneal, or parenteral routes. In other alternative embodiments,the conjugate can be administered ex vivo by direct exposure to cells,tissues or organs originating from a subject.

To formulate the pharmaceutical compositions, the conjugate can becombined with various pharmaceutically acceptable additives, as well asa base or vehicle for dispersion of the conjugate. Desired additivesinclude, but are not limited to, pH control agents, such as arginine,sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like.In addition, local anesthetics (for example, benzyl alcohol),isotonizing agents (for example, sodium chloride, mannitol, sorbitol),adsorption inhibitors (for example, Tween 80), solubility enhancingagents (for example, cyclodextrins and derivatives thereof), stabilizers(for example, serum albumin), and reducing agents (for example,glutathione) can be included. Adjuvants, such as aluminum hydroxide (forexample, Amphogel, Wyeth Laboratories, Madison, N.J.), Freund'sadjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton,Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many othersuitable adjuvants well known in the art, can be included in thecompositions. When the composition is a liquid, the tonicity of theformulation, as measured with reference to the tonicity of 0.9% (w/v)physiological saline solution taken as unity, is typically adjusted to avalue at which no substantial, irreversible tissue damage will beinduced at the site of administration. Generally, the tonicity of thesolution is adjusted to a value of about 0.3 to about 3.0, such as about0.5 to about 2.0, or about 0.8 to about 1.7.

The conjugate can be dispersed in a base or vehicle, which can include ahydrophilic compound having a capacity to disperse the conjugate, andany desired additives. The base can be selected from a wide range ofsuitable compounds, including but not limited to, copolymers ofpolycarboxylic acids or salts thereof, carboxylic anhydrides (forexample, maleic anhydride) with other monomers (for example, methyl(meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers,such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives, such as hydroxymethylcellulose,hydroxypropylcellulose and the like, and natural polymers, such aschitosan, collagen, sodium alginate, gelatin, hyaluronic acid, andnontoxic metal salts thereof. Often, a biodegradable polymer is selectedas a base or vehicle, for example, polylactic acid, poly(lacticacid-glycolic acid) copolymer, polyhydroxybutyric acid,poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such aspolyglycerin fatty acid esters, sucrose fatty acid esters and the likecan be employed as vehicles. Hydrophilic polymers and other vehicles canbe used alone or in combination, and enhanced structural integrity canbe imparted to the vehicle by partial crystallization, ionic bonding,cross-linking and the like. The vehicle can be provided in a variety offorms, including fluid or viscous solutions, gels, pastes, powders,microspheres and films for direct application to a mucosal surface.

The conjugate can be combined with the base or vehicle according to avariety of methods, and release of the conjugate can be by diffusion,disintegration of the vehicle, or associated formation of waterchannels. In some circumstances, the conjugate is dispersed inmicrocapsules (microspheres) or nanocapsules (nanospheres) prepared froma suitable polymer, for example, isobutyl 2-cyanoacrylate (see, forexample, Michael et al. J. Pharmacy Pharmacol. 43:1-5, 1991), anddispersed in a biocompatible dispersing medium, which yields sustaineddelivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain aspharmaceutically acceptable vehicles substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, and triethanolamineoleate. For solid compositions, conventional nontoxic pharmaceuticallyacceptable vehicles can be used which include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesiumcarbonate, and the like.

Pharmaceutical compositions for administering the conjugate can also beformulated as a solution, microemulsion, or other ordered structuresuitable for high concentration of active ingredients. The vehicle canbe a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof.Proper fluidity for solutions can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of a desired particlesize in the case of dispersible formulations, and by the use ofsurfactants. In many cases, it will be desirable to include isotonicagents, for example, sugars, polyalcohols, such as mannitol andsorbitol, or sodium chloride in the composition. Prolonged absorption ofthe conjugate can be brought about by including in the composition anagent which delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the conjugate can be administered in a timerelease formulation, for example in a composition which includes a slowrelease polymer. These compositions can be prepared with vehicles thatwill protect against rapid release, for example a controlled releasevehicle such as a polymer, microencapsulated delivery system orbioadhesive gel. Prolonged delivery in various compositions of thedisclosure can be brought about by including in the composition agentsthat delay absorption, for example, aluminum monostearate hydrogels andgelatin. When controlled release formulations are desired, controlledrelease binders suitable for use in accordance with the disclosureinclude any biocompatible controlled release material which is inert tothe active agent and which is capable of incorporating the conjugateand/or other biologically active agent. Numerous such materials areknown in the art. Useful controlled-release binders are materials thatare metabolized slowly under physiological conditions following theirdelivery (for example, at a mucosal surface, or in the presence ofbodily fluids). Appropriate binders include, but are not limited to,biocompatible polymers and copolymers well known in the art for use insustained release formulations. Such biocompatible compounds arenon-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects, such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-co-glycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water-soluble peptides(U.S. Pat. No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the conjugate in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theconjugate and/or other biologically active agent into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the conjugate plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, theconjugate can be delivered to a subject in a manner consistent withconventional methodologies associated with management of the disorderfor which treatment or prevention is sought. In accordance with thedisclosure herein, a prophylactically or therapeutically effectiveamount of the conjugate and/or other biologically active agent isadministered to a subject in need of such treatment for a time and underconditions sufficient to prevent, inhibit, and/or ameliorate a selecteddisease or condition or one or more symptom(s) thereof.

Typical subjects intended for treatment with the compositions andmethods of the present disclosure include humans, as well as non-humanprimates and other animals. To identify subjects for prophylaxis ortreatment according to the methods of the disclosure, accepted screeningmethods are employed to determine risk factors associated with atargeted or suspected disease of condition (for example, coughingdisease) as discussed herein, or to determine the status of an existingdisease or condition in a subject. These screening methods include, forexample, conventional work-ups to determine environmental, familial,occupational, and other such risk factors that may be associated withthe targeted or suspected disease or condition, as well as diagnosticmethods, such as various ELISA and other immunoassay methods, which areavailable and well known in the art to detect and/or characterizedisease-associated markers. These and other routine methods allow theclinician to select patients in need of therapy using the methods andpharmaceutical compositions of the disclosure. In accordance with thesemethods and principles, a conjugate and/or other biologically activeagent can be administered according to the teachings herein as anindependent prophylaxis or treatment program, or as a follow-up, adjunctor coordinate treatment regimen to other treatments, including surgery,vaccination, immunotherapy, hormone treatment, cell, tissue, or organtransplants, and the like.

The conjugates can be used in coordinate vaccination protocols orcombinatorial formulations. In certain embodiments, novel combinatorialimmunogenic compositions and coordinate immunization protocols employseparate immunogens or formulations, each directed toward eliciting ananti-LPS or an anti-LOS immune response. Separate immunogens that elicitthe anti-LPS or anti-LOS immune response can be combined in a polyvalentimmunogenic composition administered to a subject in a singleimmunization step, or they can be administered separately (in monovalentimmunogenic compositions) in a coordinate immunization protocol. Forexample, a combinatorial or a polyvalent immunogenic composition couldinclude (i) an oligosaccharide or polysaccharide obtained fromBordetella bronchiseptica or Bordetella pertussis as a first componentand (ii) oligosaccharide or polysaccharide obtained from Bordetellaparapertussis as a second component.

The administration of the conjugate of the disclosure can be for eitherprophylactic or therapeutic purpose. When provided prophylactically, theconjugate is provided in advance of any symptom. The prophylacticadministration of the conjugate serves to prevent or ameliorate anysubsequent infection. When provided therapeutically, the conjugate isprovided at (or shortly after) the onset of a symptom of disease orinfection. The conjugate of the disclosure can thus be provided prior tothe anticipated exposure to Haemophilus ducreyi, Bordetellabronchiseptica, Bordetella parapertussis, Bordetella pertussis, Vibriocholere, Shigella sp. or Haemophilus influenza, so as to attenuate theanticipated severity, duration or extent of an infection and/orassociated disease symptoms, after exposure or suspected exposure to thebacteria, or after the actual initiation of an infection.

For prophylactic and therapeutic purposes, the conjugate can beadministered to the subject in a single bolus delivery, via continuousdelivery (for example, continuous transdermal, mucosal or intravenousdelivery) over an extended time period, or in a repeated administrationprotocol (for example, by an hourly, daily or weekly, repeatedadministration protocol). The therapeutically effective dosage of theconjugate can be provided as repeated doses within a prolongedprophylaxis or treatment regimen, that will yield clinically significantresults to alleviate one or more symptoms or detectable conditionsassociated with a targeted disease or condition as set forth herein.Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby administration protocols that significantly reduce the occurrence orseverity of targeted disease symptoms or conditions in the subject.Suitable models in this regard include, for example, murine, rat,porcine, feline, non-human primate, and other accepted animal modelsubjects known in the art. Alternatively, effective dosages can bedetermined using in vitro models (for example, immunologic andhistopathologic assays). Using such models, only ordinary calculationsand adjustments are required to determine an appropriate concentrationand dose to administer a therapeutically effective amount of theconjugate (for example, amounts that are effective to elicit a desiredimmune response or alleviate one or more symptoms of a targeteddisease). In alternative embodiments, an effective amount or effectivedose of the conjugate may simply inhibit or enhance one or more selectedbiological activities correlated with a disease or condition, as setforth herein, for either therapeutic or diagnostic purposes.

The actual dosage of the conjugate will vary according to factors suchas the disease indication and particular status of the subject (forexample, the subject's age, size, fitness, extent of symptoms,susceptibility factors, and the like), time and route of administration,other drugs or treatments being administered concurrently, as well asthe specific pharmacology of the conjugate for eliciting the desiredactivity or biological response in the subject. Dosage regimens can beadjusted to provide an optimum prophylactic or therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental side effects of the conjugate and/or other biologicallyactive agent is outweighed in clinical terms by therapeuticallybeneficial effects. A non-limiting range for a therapeutically effectiveamount of a conjugate and/or other biologically active agent within themethods and formulations of the disclosure is about 0.01 mg/kg bodyweight to about 10 mg/kg body weight, such as about 0.05 mg/kg to about5 mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kg body weight.

Upon administration of a conjugate of the disclosure (for example, viainjection, aerosol, oral, topical or other route), the immune system ofthe subject typically responds to the immunogenic composition byproducing antibodies specific for LPS, LOS and/or an antigenic epitopepresented by the conjugate. Such a response signifies that animmunologically effective dose of the conjugate was delivered. Animmunologically effective dosage can be achieved by single or multipleadministrations (including, for example, multiple administrations perday), daily, or weekly administrations. For each particular subject,specific dosage regimens can be evaluated and adjusted over timeaccording to the individual need and professional judgment of the personadministering or supervising the administration of the conjugate. Insome embodiments, the antibody response of a subject administered thecompositions of the disclosure will be determined in the context ofevaluating effective dosages/immunization protocols. In most instancesit will be sufficient to assess the antibody titer in serum or plasmaobtained from the subject. Decisions as to whether to administer boosterinoculations and/or to change the amount of the composition administeredto the individual can be at least partially based on the antibody titerlevel. The antibody titer level can be based on, for example, animmunobinding assay which measures the concentration of antibodies inthe serum which bind to a specific antigen, for example, LPS and/or LOS.

Dosage can be varied by the attending clinician to maintain a desiredconcentration at a target site (for example, the lungs or systemiccirculation). Higher or lower concentrations can be selected based onthe mode of delivery, for example, trans-epidermal, rectal, oral,pulmonary, or intranasal delivery versus intravenous or subcutaneousdelivery. Dosage can also be adjusted based on the release rate of theadministered formulation, for example, of an intrapulmonary spray versuspowder, sustained release oral versus injected particulate ortransdermal delivery formulations, and so forth. To achieve the sameserum concentration level, for example, slow-release particles with arelease rate of 5 nanomolar (under standard conditions) would beadministered at about twice the dosage of particles with a release rateof nanomolar.

The methods of using conjugates, and the related compositions andmethods of the disclosure, are useful in increasing resistance to,preventing, ameliorating, and/or treating infection and disease causedby Bordetella, H. ducreyi, Vibrio cholere, Shigella sp. or Haemophilusinfluenza in animal hosts, and other, in vitro applications. Theseimmunogenic compositions can be used for active immunization forprevention of infection, and for preparation of immune antibodies. Theimmunogenic compositions are composed of non-toxic components, suitablefor infants, children of all ages, and adults.

The methods of the disclosure are broadly effective for treatment andprevention of bacterial disease and associated inflammatory, autoimmune,toxic (including shock), and chronic and/or lethal sequelae associatedwith bacterial infection. Therapeutic compositions and methods of thedisclosure for prevention or treatment of toxic or lethal effects ofbacterial infection are applicable to a wide spectrum of infectiousagents. Non-lethal toxicities that will be ameliorated by these methodsand compositions can include fatigue syndromes, inflammatory/autoimmunesyndromes, hypoadrenal syndromes, weakness, cognitive symptoms andmemory loss, mood symptoms, neurological and pain syndromes andendocrine symptoms. Any significant reduction or preventive effect ofthe conjugate with respect to the foregoing disease condition(s) orsymptom(s) administered constitutes a desirable, effective property ofthe subject composition/method of the disclosure.

The instant disclosure also includes kits, packages and multi-containerunits containing the herein described pharmaceutical compositions,active ingredients, and/or means for administering the same for use inthe prevention and treatment of bacterial diseases and other conditionsin mammalian subjects. Kits for diagnostic use are also provided. In oneembodiment, these kits include a container or formulation that containsone or more of the conjugates described herein. In one example, thiscomponent is formulated in a pharmaceutical preparation for delivery toa subject. The conjugate is optionally contained in a bulk dispensingcontainer or unit or multi-unit dosage form. Optional dispensing meanscan be provided, for example a pulmonary or intranasal spray applicator.Packaging materials optionally include a label or instruction indicatingfor what treatment purposes and/or in what manner the pharmaceuticalagent packaged therewith can be used.

The subject matter of the present disclosure is further illustrated bythe following non-limiting Examples.

Example 1 Bordetella Conjugates

Bacteria and cultivation. The following strains were obtained from ATCC:B. bronchiseptica ATCC 10580, Rb50 (ATCC BAA-588), and B. parapertussisATCC 1589; B. bronchiseptica 15374, 3145 and B. parapertussis 12822 wereobtained from Dr. M. Perry (NRC Canada). Bacteria were grown onBordet-Genguo (BG) agar plates and then transferred to Stainer-Scholte(S-S) media (Stanier D W, Scholte M J. A simple chemically definedmedium for the production of phase I Bordetella pertussis. J ClinPathol. 25:732-733, 1970). Bacteria were harvested, killed by boilingfor 1 hour and frozen for LPS extraction.

Oligosaccharides. LPS was isolated by phenol-water extraction andpurified by enzyme treatment and ultracentrifugation as described inWestphal O., Jann K., Meth. Carbohydr. Chem. 5:83-89, 1965, which isincorporated herein by reference in its entirety. To isolate O-specificoligosaccharide (O-SP), LPS (100 mg) was treated with 1% acetic acid (10ml) for 60 minutes at 100° C., ultracentrifuged and thecarbohydrate-containing supernatant was fractionated on a BioGel P-4column (1.0×100 cm) in pyridine/acetic acid/water buffer (4/8/988 ml)monitored with a Knauer differential refractometer. 28 mg of O-SP waseluted in void volume and used for conjugation. Alternatively, LPS wasdeaminated in the following way: 100 mg of LPS was dissolved in themixture: 6 ml 30% acetic acid, 6 ml 5% sodium nitrite, 6 ml water.Reaction was carried out in room temperature, 6 hours, on the magneticstirrer followed by ultracentrifugation. The supernatant was lyophilizedand purified on BioGel P-4 column using conditions as above. 23 mg ofO-SP_(deam) was eluted in void volume and used for conjugation.

Analytic. Protein concentration was measured by the method of Lowry (O.H. Lowry et al., J. Biol. Chem. 193:265, 1951). SDS-PAGE used 14% gelsaccording to the manufacturer's instructions. Double immunodiffusion wasperformed in 1.0% agarose gel in PBS.

Spectroscopy.

MALDI-TOF mass spectra of the derivatized carrier proteins and theconjugates were obtained with an OmniFlex MALDI-TOF instrument (BnikerDaltonics) operated in the linear mode. Samples for analysis weredesalted and 1 μl was mixed with 20 μl of sinapic acid matrix made in30% CH₃CN and 0.1% trifluoroacetic acid. Next, 1 μl of mixture was driedon the sample stage and placed in the mass spectrometer.

Methods. NMR spectra were recorded at 30° C. in D₂O on a Varian UNITYINOVA 500, 600, or 800 instrument, using acetone as reference for proton(2.225 ppm) and carbon (31.5 ppm) spectra. Varian standard programsCOSY, NOESY (mixing time of 400 ms), TOCSY (spinlock time 120 ms), HSQC,and gHMBC (long-range transfer delay 100 ms) were used with digitalresolution in F2 dimension <2 Hz/pt. ESI-MS and NMR spectroscopy wasused to confirm the structure of bordatellae LPS structure.

Molecular mass obtained from MALDI like 135 kDa is a mass of conjugate,from which is subtracted a mass of aminooxylated protein-likeaminooxylated-BSA is 73 kDa; the difference is a mass of oligo(poly)saccharide introduced on protein.

Conjugation.

(1) BSA-ONH₂/O-SP. An aminooxylated bovine serum albumin (BSA) wasprepared via a two-step procedure as described in Kielb et al., J. Org.Chem. 70:6987-6990, 2005, which is incorporated herein by reference inits entirety. First, the protein was treated with succinimidyl3-(bromoacetamido)propionate (SBAP) to introduce thiol-reactivebromoacetamido moieties. Next, it was coupled withO-(3-thiolpropyl)hydroxylamine, a heterobifunctional linker, to form theaminooxylated protein through stable thioether linkages (BSA-ONH₂). Forconjugation with O-SP, BSA-ONH₂ (5 mg) was reacted with 10 mg of O-SP in1.5 ml Buffer A (PBS, 0.1% glycerol, 0.005 M EDTA, pH 7.4), at pH 5.7,for 15 hours. Next, it was purified by Sephadex G100 gel filtration in0.2 M NaCl as eluant and the void volume fraction characterized byprotein and sugar assays, immunodiffusion, SDS-PAGE and MALDI-TOFspectroscopy. Three conjugates were obtained this way and named asBSA-ONH₂/Bb10580 (#1), BSA-ONH₂/BbRb50 (#2) and BSA-ONH₂/Bp15898 (#3).

(2) BSA-ONH₂/O-SP_(deam) BSA was derivatized to BSA-ONH₂ as above and 5mg was reacted with 10 mg of O-SP_(deam) using the same condition asabove. Next, it was purified by Sephadex G100 gel filtration and assayedas above. The products were named BSA-ONH₂/Bb10580_(deam) (#4),BSA-ONH₂/BbRb50_(deam) (#5) and BSA-ONH₂/Bp15898_(deam) (#6).

Immunization. 5 to 6-weeks-old female NIH Swiss Webster mice wereimmunized s.c. 3 times at 2 weeks intervals with 2.5 μg O-SP as aconjugate in 0.1 ml PBS and groups of 10 exsanguinated 7 days after thesecond or third injections. Controls received PBS. Hyperimmune mice seraagainst B. bronchiseptica strains 10580 and Rb50, and against B.parapertussis strain 15898 were induced by multiple intraperitronealimmunization of mice with heat killed whole bacterial cells.

Antibodies. Serum IgG antibodies were measured by ELISA. Nunc Maxisorbplates were coated with B. bronchiseptica 10580 LPS, Rb50LPS or B.parapertussis 15898 LPS at 5 μg/ml in PBS containing 1% trichloroaceticacid as described in Hardy et al., “Enhanced ELISA sensitivity using TCAfor efficient coating of biologically active lipopolysaccharides orlipid A to the solid phase,” J. Immunol Methods 176(1):111-6, 1994.Concentration and the composition of buffer for coating antigen weredetermined by checkerboard titration. Plates were blocked with 1% BSA inPBS for hyperimmune sera or 1% HSA in PBS for conjugate-induced sera for1 hour at room temperature. A MRX Dynatech reader was used. Antibodylevels were calculated relative to hyperimmune standard serum diluted1:20,000 for B. bronchisepaca 10580; 1:15,000 for B. bronchiseptica Rb50and 1:10,000 for B. parapertussis 15898 and assigned a value of 1000ELISA units (EU). Results were computed with an ELISA data processingprogram provided by the Biostatistics and Information Management Branch,CDC.

Inhibition ELISA was done by incubating hyperimmune mice sera, dilutedto the concentration that gave an A₄₀₅ absorption of 1.0, with 10 or 50μg of inhibitor per well, for 1 hour at 37° C. and overnight at 4° C.The assay was then continued as above. Sera with and without inhibitor,at the same dilution, were compared. Percent inhibition was defined as(1-A₄₀₅ adsorbed serum/A₄₀₅ non-adsorbed serum)×100%.

The results are shown below in Table 1.

TABLE 1 Inhibition ELISA. O-SP Amidation end- of second % of inhibitionof hyperimmune whole cell sera Inhibitor group O-SP sugar Anti-Bb10580Anti-BbRb50 Anti-Bp15989 Bb 10580 O-SP Ala − 92 2 50 Bb 110H O-SP Ala −96 3 45 Bb Rb50, O-SP Lac − 0 93 1 Bb 512 O-SP Lac − 1 95 3 Bp 15898O-SP Ala + 42 5 97 Bp 15311 O-SP Ala + 50 3 98 H. ducrei O-SP na Na 0 00 Plates were coated with B. bronchiseptica 10580 LPS, Rb50 or B.parapertussis 15989, respectively at 5 μg/ml and reacted anti-B.bronchiseptica 10580 hyperimmune mice serum diluted 1:40000, Rb501:20000 and anti-B. parapertussis 15989 1:20000. Inhibitors were used at50 μg/well

Results. Chemical Characterization of LPSes

Chemical analysis indicated that strains B. bronchiseptica 10580, 15374and 5137, as well as strains B. parapertussis 15898 and 12822 belong tothe “Ala-type” (terminal non-reducing residue-2,3,4-triamino-2,3,4-trideoxy-alpha-galactouronamide is formylated onposition 3 and 4 and has N-formyl-L-alanyl or L-alanyl substituents atN-2), whereas strain B. bronchiseptica Rb50 belongs to the “Lac-type”(the same terminal residue is acetylated on position 2, formylated atposition 3 and the amino group at position 4 bears a 2-methoxypropionylsubstituent).

Serum Antibodies.

Immunogenicity was checked by injection to mice. The average molecularmass of deaminated O-SP from B. bronchiseptica strain 10580, as assayedby ES-MS, was established as 5108 Da. The number of O-SP chains boundper one BSA was estimated to be 15 in conjugate #4. The average mass ofO-SP was calculated on the bases of detailed structural analysis ofstudies LPSes, as reported elsewhere, was established as average of 6588Da for conjugate #1, 2 and 3. The number of O-SP chains bound per oneBSA was estimated to be 10, 10 and 15, respectively. The result areshown below in Table 2.

TABLE 2 Composition and serum GM of IgG anti-B. bronchiseptica and B.parapertussis LPS in mice by conjugates of O-SP and O-SP_(deam) bound tobovine serum albumin (BSA). Mol. Ratio Mol O- ELISA units after 3rdinjection mass¹ protein/ SP/Mol Coating antigen # Conjugate [kDa] sugarProtein 10580 LPS Rb50 LPS 15898 LPS 1 BSA-ONH₂/Bb10580 135 1:0.9 9 4.90.3 0.4 2 BSA-ONH₂/BbRb50 137 1:0.9 9 2.4 132 0.4 3 BSA-ONH₂/Bp15898 1651:1.4 14 0.3 0.3 12 4 BSA-ONH₂/Bb10580_(deam) 130 1:1.0 11 55 0.6 4.8 5BSA-ONH₂/BbRb50_(deam) 105 1:0.5 8 0.1 3.5 0.3 6 BSA-ONH₂/Bp15898_(deam)116 1:0.7 10 0.5 0.1 15.6 Mice (10 per group) were immunized with 2.5 μgof polysaccharide as a conjugate/mouse, injected s.c., 3 times, 2 weeksapart. ¹Mol mass was assayed by Maldi-tof, Mol mass of BSA-ONH₂ was 74.2kDa The “Ratio protein/sugar” is the mass ratio of the two components ofthe final conjugate. The “Mol O-SP/Mol Protein is the mole ratio of thetwo components of the final conjugate.

A novel pentasaccharide was identified in B. bronchiseptica and B.parapertussis LPS. B. bronchiseptica O-SP differed in their non-reducingend-groups: the “ala-type” and the “lac-type.” In contrast, all B.parapertussis strains analyzed belonged to the “ala-type.” No crossreaction between the two types of B. Bronchiseptica LPS was observed.Inhibition assays showed that the terminal residues of O-SP areimmunodominant. BSA/O-SP conjugates were specific and induced antibodiesonly to the homologous type of O-SP. Accordingly, and based uponepidemiological data, at least two types of LPS should be included in avaccine according to a preferred embodiment.

Example 2 Haemophilia ducreyl Conjugates

Bacteria and cultivation. Haemophilus ducreyi strains 35000 was obtainedfrom Culture Collection Göteborg University (CCUG 7470). Bacteria werecultivated on chocolate agar plates Grand Lux (GLV-3) (Department ofBacteriology, Sahlgrenska Hospital, Göteborg, Sweden), containing 5%brain heart infusion (BHI) agar, 1% horse blood, 1.5% horse serum, 0.06%yeast autolysate, 0.015% IsoVitalex (BBL) and 3 mg/ml vancomycin. Theplated bacteria were incubated at 33° C. for 48 hours in high humidityin an anaerobic jar with Anaerocult C (Merk, Darmstad, Germany) forgeneration of an oxygen-depleted and CO₂ enriched atmosphere. Thebacteria were harvested and frozen at −20° C. for LOS extraction.

Oligosaccharides. LOS was isolated by phenol-water extraction andpurified by enzyme treatment and ultracentrifugation as described inWestphal et al., Meth. Carbohydr. Chem. 5:83-89, 1965. To isolateoligosaccharide (OS), LOS (100 mg) was treated with 1% acetic acid (10ml) for 60 minutes at 100° C. and the carbohydrate-containingsupernatant was fractionated on a BioGel P-4 column (1.0×100 cm) in 0.05M pyridine acetate buffer (pH 5.6) and monitored with a Knauerdifferential refractometer.

Analytic. Protein concentration was measured by the method of Lowry(Lowry et al., J. Biol. Chem. 193:265, 1951), sugar concentration byphenol/H₂SO₄ assay (Dubois et al., “Colorimetric method fordetermination of sugars and related substances,” Anal. Chem.,28:350-356, 1956), incorporation of benzaldehyde groups by colorimetricreaction with 2-hydrazinopyride (Solulink protocol), and hydrazide byTNBS assay as reported (Habeeb A F, “Determination of free amino groupsin proteins by trinitrobenzenesulfonic acid,” Anal Biochem.14(3):328-336, 1966).

Spectroscopy. Sugars were analyzed according to Sawardeker et al(Sawardeker et al., “Quantitative determination of monosaccharides astheir alditol acetates by gas-liquid chromatography,” Analyt. Chem37:1602-1604, 1965). A 0.5 mg sample of each polysaccharide washydrolyzed in 1 ml of 10 M HCl for 30 minutes at 80° C., reducedperacetylated and analyzed by GLC-MS using Hewlett-Packard apparatus,model HP 6890, with a type HP-5 glass capillary column (0.32 mm×30 m)and temperature programming at 8° C./minute, from 125-250° C. in theelectron ionization (106 eV) mode. Methylation was performed asdescribed in Ciucanu et al., “A simple and rapid method for thepermethylation of carbohydrates,” Carbohydr. Res. 131:209-217, 1984.Methylated compounds were hydrolyzed, converted to alditol acetates, andanalyzed by GLC-MS as above. MALDI-TOF mass spectra were obtained withan OmniFlex MALDI-TOF instrument (Bruker Daltonics) operated in thelinear mode. Samples for analysis were desalted and 1 μl was mixed with20 μl of sinnapinic acid matrix made in 30% CH₃CN and 0.1%trifluoroacetic acid. Next, 1 μl of mixture was dried on the samplestage and placed in the mass spectrometer. ESI-MS spectra were recordingon the Agilent Series LC/MSD instrument in the negative ion mode ¹H, ¹³Cand ³¹P NMR spectra were recorded at 300 MHz using Varian spectrometer.Solutions of 5-13 mg of analytats in D₂O (99.96 atom % D) were used,with acetone as an internal reference at 2.225 ppm and 31.0 ppm, for ¹Hand ¹³C respectively, or 85% H₃PO₄ containing 10% D₂O as an externalreference for ³¹P at −0.73 ppm.

Conjugation.

Conjugation by oxime formation. Aminooxylated BSA or Tetanus toxoid (TI)was prepared via a two step procedure as described in Kielb et al., J.Org. Chem. 70:6987-6990, 2005, which is incorporated herein by referencein its entirety. First, the protein was treated with succinimidyl3-(bromoacetamido)propionate (SBAP) to introduce thiol-reactivebromoacetamido moieties. Next, it was coupled withO-(3-thiolpropyl)hydroxylamine, a heterobifunctional linker featuringterminal aminooxy and thiol groups, to form the aminooxylated proteinthrough stable thioether linkages (Pr—ONH₂). For conjugation with O-SP,Pr—ONH₂ (10 mg) was reacted with 10 mg of O-SP in 1.5 ml Buffer A (PBS,0.1% glycerol, 0.005 M EDTA, pH 7.4), at pH 5.7, for 15 hours. Next, itwas purified by Sephadex G100 gel filtration in 0.2 M NaCl as eluant andthe void volume fraction characterized by protein and sugar assays,immunodiffusion, SDS-PAGE and MALDI-TOF spectroscopy. Two conjugateswere obtained this way and named as BSA-ONH₂/OS (#1) and TT-ONH₂/OS(#2).

Characterization of H. ducreyi oligosaccharide epitpopes in theconjugates by monoclonal antibodies. The maxi-sorp ELISA plates werecoated with the conjugates (1-2) in concentration 2 μg/ml of sugar as aconjugate and 10 μg/ml LOS over night. As a negative control BSA (10μg/ml) was used. Plates were blocked with 1% BSA and the medium(concentrated 5×) containing monolonal antibodies (MAHD6 or MADH7 [v])was added. Plates were incubated 3 hours, washed and anti-mouse alkalinephosphatase conjugate was added. After further incubation, plates werewashed and developed. The absorbance at 403 nm was monitored.

Oxime formation with hemiacetal groups. D-Glucose (10 mg), D-maltose (10mg) maltotriose (25 mg), D-glucosamine (10 mg) or N-acetyl-D-mannosamine(10 mg) were reacted with O-(3-thiolpropyl)hydroxylamine (6 mg) in 1 mlD₂O adjusted to pH 5.5 with 30% solution of NaOD at 37° C. for 15 hours.Progress of reaction was monitored by ¹H NMR. Maltotriose-SH (30 mg) wasseparated from linker by passing through BioBel P-2 column in 0.05 Mpyridine acetate buffer as above and freeze-dried. Next 30 mg ofmaltotriose-SH was reacted with bromoacetamido-derivatized BSA (15 mg),prepared as above to form maltotriose-BSA conjugate by thioetherlinkages. Reaction was done in buffer A, pH 7.4, 3 hours and solutionwas purified on Sephadex G-100 column as above. Extent of conjugationwas evaluated by MALDI-TOF. Molecular mass of bromoacetamido-BSA was73545 Da, while maltotriose-BSA conjugate was 81673 Da, indicatedincorporation of 16 maltotriose molecules per BSA.

Immunization. 5 to 6-weeks-old female NIH Swiss Webster mice wereimmunized sc 3 times at 2 weeks intervals with 2.5 μg OS or PGA as aconjugate in 0.1 ml PBS and groups of 10 exsanguinated 7 days after thesecond or third injections. Controls received PBS.

Antibodies. Serum IgG antibodies were measured by ELISA (Taylor et al.,Infect. Immun. 61:3678-3687, 1993). Nunc Maxisorb plates were coatedwith H. ducreyi LOS, 10 μg/ml PBS (determined by checkerboardtitration). A MRX Dynatech reader was used. The reference serum to O-SPand BSA was a pool of sera obtained from mice immunized 3 times with 5μg of oligosaccharide as a conjugate BSA-CHO/AH-O-SP (Conj. #3), dilutedto 1.2000 in the first well and assigned a value of 1000 ELISA units(EU). The reference serum to TT was a pool of sera obtained from miceimmunized 3 times with 5 μg of oligosaccharide as a conjugateTT-NOS/O-SP (Conj. #2), diluted to 1:5000 in the first well and assigneda value of 100 ELISA units (EU). Results were computed with an ELISAdata processing program provided by the Biostatistics and InformationManagement Branch, CDC.

Immunology. SDS-PAGE and Western-blotting used 14% gels according to themanufacturer's instructions. Double immunodiffusion was performed in1.0% agarose gel in PBS.

Results

Characterization of LOS and LOS-derived oligosaccharides. Massspectroscopic and NMR analysis of isolated products confirmed thestructure of the sugar chain of H. ducreyi strain 35000 LOS. The datawere in agreement with the published structure (Melaugh et al.,“Structure of the major oligosaccharide from the lipooligosaccharide ofHaemophilus ducreyi strain 35000 and evidence for additionalglycoforms,” Biochemistry 33(44):13070-13078, 1994) as representedbelow:

The sialilation of non-reducing end was estimated at about 60% by NMRand GLC-MS analysis. Hydrolysis of LOS with 1% acetic acid cleaved O-SPfrom Lipid A on the KDO residue, removing at the same time all sialicacid residues from non-reducing end. The observed molecular mass of thisproduct, recorded by ESI-MS in negative mode was [M−1]=1675.8, which isin agreement with the structure of Hex₃HexNAcHep₄-anhydro-Kdo as theO-SP for H. ducreyi strain 35000. No phosphate group on KDO wasdetectable also by ³¹P-NMR suggested the beta-elimination of phosphatefrom KDO as was reported that results in anhydro-KDO groups at thereducing end (Auzanneau et al., “Phosphorylated sugars. Part 27.Synthesis and reactions, in acid medium, of 5-O-substituted methyl3-deoxy-α-D-manno-oct-2-ulopyranosic acid 4-phosphates,” J. Chem. Soc.Perkin Transl. 1:509-517, 1991; Vinogradov et al., “The structure of thecarbohydrate backbone of the core-lipid-A region of thelipopolysaccharide from Vibrio cholerae strain H11 (non-01),” Eur JBiochem. 218(2):543-554, 1993). A reactive ketone group was shown toform during beta elimination of a model compound,5-O-methyl-KDO-4-phosphate. The ketone group was used for conjugation ofthe OS to the protein carrier.

Characterization of conjugates. Conjugation of O-SP to aminooxylatedprotein gave a conjugate containing average 15 chains of oligosaccharideper protein (#1 and #2). Protein/sugar ratio was analyzed bycolorimetric assays and by increase of molecular mass using MALDI-TOFspectroscopy. Although not bound by any theory, it is believed that theconjugate is formed by the reaction of a ketone group on the terminalKDO molecule with O-alkyl hydroxylamine on the protein.

In order to identify the core structure of the H. ducreyi LOS in theconjugates two monoclonal antibodies were used to structurally definedepitopes on the H. ducreyi LOS [V]

Conjugates #1 and 2 showed similar levels of recognition as LOS itself(see Table 3 below).

TABLE 3 Binding (ELISA) of Mabs specific to H. ducreyi LOS withOS-protein conjugates. Plates were coated with conjugates and reactedwith Mabs. Absorbance at 403 nm Conjugates Mab MAHD6 MAHD7 BSA-ONH₂/OS2.73 4.18 TT- ONH₂/OS 1.86 3.95 LOS 1.77 3.6 BSA 0.08 0.08

Immunology. The conjugates were injected into mice at a dose of 2.5 or 5microgram of OS as a conjugate per mouse and the IgG anti-H. ducreyi LOSlevels were assayed by ELISA. The results are presented in Table 4below. Negligible levels of anti-LOS antibodies were detected in sera.However, when plates were coated with conjugate #2, high level ofantibodies was detected in sera induced by conjugate #1. This means thatthe conjugates induce antibodies to sugar part of this LOS while it ispresented on other carrier protein in ELISA assay. Since carrierproteins are different, the antibodies seem to be induced against eithercommon sugar part or the linker moiety. It indicated that epitopespresented on ELISA plates by coating with LOS is different then bycoating with conjugate.

TABLE 4 Composition and serum GM of IgG anti-H. ducreyi LOS in mice byconjugates of O-SP bound to bovine serum albumin (BSA), and tetanustoxoid (TT) and by lacto-N-neotetraose and sialyl-lacto-N-neotetraosebound to human serum albumine (HSA). Ratio Mol Mol. protein/ OS/MolMicrogr. Pr/ Anti- Anti- Ani- # Conjugate mass¹ sugar Protein OSinjected LOS Protein conjugate 1 BSA-ONH₂/OS 105 kDa 2:1 18  5/2.5 2 656 29 (#2) 2 TT-ONH₂/OS Nd 6:1 15 15/2.5 4 2297 295 (#1) Mice (10 pergroup) were immunized with 2.5 μg of oligosaccharide as aconjugate/mouse and injected s.c., 3 times, 2 weeks apart. ¹assayed byMALDI-TOF The H. ducreyi OS/protein conjugate had limited immunogenicityin mice.

Example 3 B. pertussis and B. bronchiseptica Conjugates Methods:

Bacteria and cultivation. B. pertussis ATCC BAA-589 (Tohama I) and B.bronchiseptica ATCC 10580 were grown on Bordet-Gengou (BG) agar platesand transferred to Stainer-Scholte (S-S) media. Bacteria were harvestedand killed with 1% formalin.

Oligosaccharides. LPS was isolated by hot phenol-water extraction andpurified by enzyme treatment and ultracentrifugation. To isolate coreoligosaccharide (OS), LPS was treated with 1% acetic acid at 10 mg/mlfor 60 min at 100° C., ultracentrifuged and the carbohydrate-containingsupernatant fractionated on a 1.0×100 cm column of BioGel P-4 inpyridine/acetic acid/water buffer (4/8/988 ml) monitored with a Knauerdifferential refractometer.

Conjugation. BSA-ONH₂/OS. Bovine serum albumin (BSA, Sigma, St. Louis,Mo.) was derivatized to aminooxylated derivatives in a two stepprocedure as described in Kielb et al., J. Org. Chem. 70:6987-6990,2005, which is incorporated herein by reference in its entirety: (1) BSAwas treated with succinimidyl 3-(bromoacetamido)propionate (SBAP,Pierce, Pittsburgh, Pa.) to introduce thiol-reactive bromoacetamidomoieties (BSA-Br); (2) BSA-Br was coupled withO-(3-thiopropyl)hydroxylamine, a heterobifunctional linker, to form theaminooxylated protein through stable thioether linkages (BSA-ONH₂). Forconjugation with OS, BSA-ONH₂ (5 mg) was reacted with 7 mg of OS in 1.5ml Buffer A (PBS, 0.1% glycerol, 5 mM EDTA), at pH 5.7, for 15 hours.Next, it was passed through a 1×100 cm Sephadex G-50 column in 0.2 MNaCl as eluent and the void volume fraction characterized by proteinassay, immunodiffusion, SDS-PAGE and MALDI-TOF spectroscopy. Theobtained conjugates were named BSA-ONH₂/Bp (#1), BSA-ONH₂/B. b-core (#2)

Immunization was performed as described above in Example 1.

Results:

Oligosaccharides: B. pertussis LPS contains only a core region composedof 12 sugars:

B. bronchiseptica LPS contains the same core structure as B. pertussisbut it could be further substituted by O-specific chains. For this studyonly free core, with no O-SP was used, after separation on BioGel P-4column. First fraction eluted from the column contained core substitutedwith O-SP and second fraction contains free core used in this study.ESI-MS (FIGS. 6 and 7) and NMR analysis confirmed the above structurewith small variations in case of B. bronchiseptica: the methylation ofFuc4NMe is only 50% (2280 kDa pick), while in B. pertussis is 100% andHep is phosphorylated in about 30% (2374 Da pick), while in B. pertussisHep is not phosphorylated.

Conjugates. SDS-PAGE gel and Maldi analysis showed increase in molecularmass of both conjugates to average 94 kDa comparing to BSA-ONH₂ 71 kDa.Since the mass of OS is 2295 Da, the increase indicates averageincorporation of 10 OS chains per one BSA molecule in both cases.

Both conjugates reacted with anti-B. pertussis hyperimmune serum andanti-BSA serum with an observed line of identity. Both conjugatesinduced serum antibody responses on a similar level as assayed by ELISAagainst B. pertussis LOS.

Example 4 S. flexnarii 2a Conjugates Methods:

Bacteria and cultivation. Shigella flexneri type 2a strain 2457T wasgrown in ultrafiltered Triptic Soy Broth (Difco Laboratories) with 5 gof glucose and 5 mM magnesium sulphate per liter, for 20 h at 20° C.with stirring and aeration; the pH was maintained at ˜7.5 by addition ofammonium hydroxide. The identity of bacteria was confirmed by culture,Gram staining and agglutination with typing antisera. LPS was extractedby hot phenol method and after dialysis recovered from each phase.

Oligosaccharides. The LPSs (20-80 mg) were treated with 1% acetic acidat 100° C. for 1 h, precipitate of lipid A removed by centrifugation,O-specific chain (O-SP) was separated by gel chromatography on SephadexG-50 column.

Conjugation. BSA-ONH₂/OS. Bovine serum albumin (BSA, Sigma, St. Louis,Mo.) was derivatized to aminooxylated derivatives in a two stepprocedure as described in Kielb et al., J. Org. Chem. 70:6987-6990,2005, which is incorporated herein by reference in its entirety. (1) BSAwas treated with succinimidyl 3-(bromoacetamido)propionate (SBAP,Pierce, Pittsburgh, Pa.) to introduce thiol-reactive bromoacetamidomoieties (BSA-Br); (2) BSA-Br was coupled withO-(3-thiopropyl)hydroxylamine, a heterobifunctional linker, to form theaminooxylated protein through stable thioether linkages (BSA-ONH₂). Forconjugation with OS, BSA-ONH₂ (1 mg) was reacted with 3 mg of O-SP in0.3 ml Buffer A (PBS, 0.1% glycerol, 5 mM EDTA), at pH 5.7, for 15hours. Next, it was passed through a 1×100 cm Sephadex G-50 column in0.2 M NaCl as eluent and the void volume fraction characterized byprotein assay, immunodiffusion and SDS-PAGE. The obtained conjugateswere named BSA-ONH₂/Sf-OSP.

Results:

O-SP: S. flexneii O-SP contains a core region composed of 10 sugarssubstituted with a repeating unit:

Core Region:

PPE-phosphoethanolamine; RU-repeating unit

Repeating unit (about 5-15 repeats)

Conjugates. SDS-PAGE gel analysis showed increase in molecular mass of aconjugate comparing to BSA-ONH₂ to about 250 kDa. The obtained conjugatereacted with anti-S. flexnerii 2a hyperimmune serum and anti-BSA serumwith an observed line of identity.

In view of the many possible embodiments to which the principles of thedisclosed conjugates and methods may be applied, it should be recognizedthat the illustrated embodiments are only preferred examples and shouldnot be taken as limiting the scope of the invention.

1. A method for preparing an oligosaccharide-protein carrier immunogenic conjugate or polysaccharide-protein carrier immunogenic conjugate, comprising: obtaining an oligosaccharide or polysaccharide having an anhydro 3-deoxy-D-manno-octulsonic acid moiety located at the terminal reducing end of the oligosaccharide or polysaccharide that includes a carbonyl functional group; and reacting the carbonyl functional group of the anhydro 3-deoxy-D-manno-octulsonic acid moiety with an aminooxylated protein carrier molecule resulting in an oligosaccharide-protein carrier immunogenic conjugate or polysaccharide-protein carrier immunogenic conjugate that includes a covalent oxime bond between the oligosaccharide and the protein carrier or the polysaccharide and the protein carrier.
 2. The method of claim 1, wherein the oligosaccharide or polysaccharide is obtained from Haemophilus ducreyi, Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis, Vibrio cholere, Shigella sp., Haemophilus influenza, or a mixture thereof.
 3. The method of claim 1, wherein the oligosaccharide or polysaccharide is obtained from at least one bacteria containing a lipopolysaccharide with a 3-deoxy-D-manno-octulsonic acid moiety.
 4. The method of claim 1, wherein obtaining the oligosaccharide or polysaccharide comprises: isolating a lipopolysaccharide from at least one type of bacteria, wherein the lipopolysaccharide includes a Lipid A domain and at least one polysaccharide or oligosaccharide domain, the Lipid A domain and the polysaccharide or oligosaccharide domain being joined together by 3-deoxy-D-manno-octulsonic acid; and cleaving the Lipid A from the polysaccharide or oligosaccharide domain such that the 3-deoxy-D-manno-octulsonic acid remains linked to the polysaccharide or oligosaccharide domain.
 5. The method of claim 4, wherein cleaving the Lipid A from the polysaccharide or oligosaccharide domain comprises acid hydrolyzing the lipopolysaccharide under conditions sufficient for severing a glycosidic bond between the 3-deoxy-D-manno-octulsonic acid and the Lipid A domain.
 6. The method of claim 4, wherein cleaving the lipid A from the polysaccharide or oligosaccharide domain comprises treating the lipopolysaccharide with acetic acid.
 7. The method of claim 1, wherein the carbonyl functional group is a ketone.
 8. The method of claim 1, wherein the mol ratio of the carbonyl functional group on the oligosaccharide or polysaccharide:aminooxy on the protein carrier ranges from about 0.3:1 to about 1:3.
 9. The method of claim 1 wherein the aminooxylated protein carrier is prepared by treating a protein with at least one agent that introduces at least one aminooxy functional group onto the protein.
 10. The method of claim 9 wherein the aminooxy-introducing agent is selected from aminoooxy-alkyl-thiol and aminoooxy-aryl-thiol.
 11. The method of claim 9, further comprising treating the protein with a treatment agent that introduces at least one thiol-reactive group onto the protein prior to treating the protein with the aminooxy-introducing agent.
 12. The method of claim 11, wherein the thiol-reactive group is a haloacetamido moiety.
 13. The method of claim 1, wherein the oligosaccharide or polysaccharide is obtained from Bordetella bronchiseptica, Bordetella parapertussis, or Bordetella pertussis.
 14. The method of claim 1, wherein the oligosaccharide or polysaccharide is obtained from at least one bacteria containing a lipopolysaccharide with a 3-deoxy-D-manno-octulsonic acid moiety phosphorylated at position C4 on the 3-deoxy-D-manno-octulsonic acid moiety.
 15. The method of claim 1, wherein the oligosaccharide or polysaccharide is obtained from Shigella flexneri.
 16. An immunogenic conjugate prepared according to claim
 1. 17. An immunogenic conjugate comprising the structure of: Pr—Sp—O—N═C(COOH)—anh-KDO—OS wherein Pr is a carrier protein, Sp is an optional spacer moiety, anh-KDO is an anhydro moiety from 3-deoxy-D-manno-octulsonic acid, and OS is an oligosaccharide or polysaccharide residue from the cleavage of Lipid A from a lipopolysaccharide.
 18. The conjugate of claim 17, wherein the lipopolysaccharide is obtained from Haemophilus ducreyi, Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis, Vibrio cholere, Shigella sp., Haemophilus influenza, or a mixture thereof.
 19. The conjugate of claim 17, wherein the oligosaccharide or polysaccharide includes an O-antigen chain and a core oligosaccharide.
 20. The conjugate of claim 17, wherein the lipopolysaccharide is obtained from Bordetella bronchiseptica or Bordetella parapertussis and the OS includes at least: -4-β-ManNAc3ANcAN-4-β-GlcNAc3NAcAN-4-α-GalNAc-4-β-ManNAc3NAcA-3-β-FucNAc4NMe-.
 21. The conjugate of claim 17, wherein the lipopolysaccharide is obtained from Haemophilus ducreyi and the OS includes at least: Hex₃HexNAcHep₄.
 22. The conjugate of claim 17, wherein the oligosaccharide or polysaccharide is obtained from Bordetella bronchiseptica, Bordetella parapertussis, or Bordetella pertussis.
 23. The conjugate of claim 17, wherein the oligosaccharide or polysaccharide is obtained from Bordetella bronchiseptica or Bordetella pertussis.
 24. The conjugate of claim 17, wherein the oligosaccharide or polysaccharide is obtained from Bordetella parapertussis.
 25. The conjugate of claim 17, wherein the lipopolysaccharide is obtained from Bordetella pertussis and the OS includes at least:


26. The conjugate of claim 17, wherein the lipopolysaccharide is obtained from Shigella flexneri and the OS includes at least:

wherein PPE is phosphoethanolamine; and RU is a repeating unit which repeats 5 to 15 times and has a structure of:


27. A method of eliciting an immune response in a subject, comprising administering to the subject the conjugate of claim 17, thereby eliciting an immune response in the subject.
 28. A pharmaceutical composition comprising the conjugate of claim 17 and a pharmaceutically acceptable carrier.
 29. A method for preparing an oligosaccharide-protein carrier immunogenic conjugate or polysaccharide-protein carrier immunogenic conjugate, comprising: obtaining an oligosaccharide or polysaccharide having an anhydro 3-deoxy-D-manno-octulsonic acid moiety located at the terminal reducing end of the oligosaccharide or polysaccharide; reacting the anhydro 3-deoxy-D-manno-octulsonic acid moiety of the oligosaccharide or polysaccharide with a heterobifunctional compound that includes at least one aminooxy group; and then reacting the oligosaccharide or polysaccharide with a protein carrier resulting in an oligosaccharide-protein carrier immunogenic conjugate or polysaccharide-protein carrier immunogenic conjugate that includes a covalent oxime bond between the oligosaccharide and the protein carrier or the polysaccharide and the protein carrier. 